This application is a national stage filing under 35 U.S.C. 371 of PCT application PCT/SE01/01052, filed May 14, 2001, which claims priority from Sweden Application No. 0001803-6, filed May 16, 2000, the specifications of each of which are incorporated by reference herein. PCT Application PCT/SE01/01052 was published under PCT Article 21(2) in English.
This invention relates to novel pharmaceutically useful compounds, in particular compounds that are, or are prodrugs of, competitive inhibitors of trypsin-like serine proteases, especially thrombin, their use as medicaments, pharmaceutical compositions containing them and synthetic routes to their production.
Blood coagulation is the key process involved in both haemostasis (i.e. the prevention of blood loss from a damaged vessel) and thrombosis (i.e. the formation of a blood clot in a blood vessel, sometimes leading to vessel obstruction).
Coagulation is the result of a complex series of enzymatic reactions. One of the ultimate steps in this series of reactions is the conversion of the proenzyme prothrombin to the active enzyme thrombin.
Thrombin is known to play a central role in coagulation. It activates platelets, leading to platelet aggregation, converts fibrinogen into fibrin monomers, which polymerise spontaneously into fibrin polymers, and activates factor XIII, which in turn crosslinks the polymers to form insoluble fibrin. Furthermore, thrombin activates factor V and factor VIII leading to a xe2x80x9cpositive feedbackxe2x80x9d generation of thrombin from prothrombin.
By inhibiting the aggregation of platelets and the formation and crosslinking of fibrin, effective inhibitors of thrombin would be expected to exhibit antithrombotic activity. In addition, antithrombotic activity would be expected to be enhanced by effective inhibition of the positive feedback mechanism.
Further, it is known that administration of prodrugs of thrombin inhibitors may give rise to improvements in:
(a) certain pharmacokinetic properties after administration of; and
(b) the prevalence of certain side effects associated with, those inhibitors.
The early development of low molecular weight inhibitors of thrombin has been described by Claesson in Blood Coagul. Fibrinol. (1994) 5, 41 1.
Blombxc3xa4ck et al (in J. Clin. Lab. Invest. 24, suppl. 107, 59, (1969)) reported thrombin inhibitors based on the amino acid sequence situated around the cleavage site for the fibrinogen  chain. Of the amino acid sequences discussed, these authors suggested the tripeptide sequence Phe-Val-Arg (P9-P2-P1, hereinafter referred to as the P3-P2-P1 sequence) would be the most effective inhibitor.
Thrombin inhibitors based on dipeptidyl derivatives with an -aminoalkyl guanidine in the P1-position are known from U.S. Pat. No. 4,346,078 and International Patent Application WO 93/11152. Similar, structurally related, dipeptidyl derivatives have also been reported. For example International Patent Application WO 94/29336 discloses compounds with, for example, aminomethyl benzamidines, cyclic aminoalkyl amidines and cyclic aminoalkyl guanidines in the P1-position (International Patent Application WO 97/23499 discloses prodrugs of certain of these compounds); European Patent Application 0 648 780, discloses compounds with, for example, cyclic aminoalkyl guanidines in the P1-position.
Thrombin inhibitors based on peptidyl derivatives, also having cyclic aminoalkyl guanidines (e.g. either 3- or 4- aminomethyl-1-amidino-piperidine) in the P1-position are known from European Patent Applications 0 468 231, 0 559 046 and 0 641 779.
Thrombin inhibitors based on tripeptidyl derivatives with arginine aldehyde in the P1-position were first disclosed in European Patent Application 0 185 390.
More recently, arginine aldehyde-based peptidyl derivatives, modified in the P3-position, have been reported. For example, International Patent Application WO 93/18060 discloses hydroxy acids, European Patent Application 0 526 877 des-amino acids, and European Patent Application 0 542 525 O-methyl mandelic acids in the P3-position.
Inhibitors of serine proteases (e.g. thrombin) based on electrophilic ketones in the P1-position are also known. For example, European Patent Application 0 195 212 discloses peptidyl -keto esters and amides, European Patent Application 0 362 002 fluoroalkylamide ketones, European Patent Application 0 364 344 ,,-triketocompounds, and European Patent Application 0 530 1675 -alkoxy ketone derivatives of arginine in the P1-position.
Other, structurally different, inhibitors of trypsin-like serine proteases based on C-terminal boronic acid derivatives of arginine and isothiouronium analogues thereof are known from European Patent Application 0 293 881.
More recently, thrombin inhibitors based on peptidyl derivatives have been disclosed in European Patent Application 0 669 317 and International Patent Applications WO 95/35309, WO 95/23609, WO 96/25426, WO 97/02284, WO 97/46577, WO 96/32110, WO 96/31504, WO 96/03374, WO 98/06740, WO 97/49404 and WO 99/29664. Certain prodrugs of thrombin inhibitors have been disclosed in WO 97/33576.
WO 98/57932 and WO 00/35869 disclose thrombin inhibitors, and prodrugs of thrombin inhibitors, based on peptidyl derivatives with fused, bi- or tri-cyclic acids at the P3-position.
However, there remains a need for effective inhibitors of trypsin-like serine proteases, such as thrombin. There is, also a need for compounds that have a favourable pharmacokinetic profile (e.g. low clearance), are orally bioavailable, and are selective in inhibiting thrombin over other serine proteases, in particular those involved in haemostatis. Compounds which exhibit competitive inhibitory activity towards thrombin would be expected to be especially useful as anticoagulants and therefore in the therapeutic treatment of thrombosis and related disorders.
According to the invention there is provided compounds of formula I, 
wherein
Y represents S(O) or S(O)2;
R1 represents halo; and
R2 represents H, halo or C1-4 alkoxy (which latter group is optionally substituted by one or more halo groups);
or a pharmaceutically acceptable derivative thereof, which compounds are referred to hereinafter as xe2x80x9cthe compounds of the inventionxe2x80x9d.
The term xe2x80x9cpharmaceutically acceptable derivativesxe2x80x9d of compounds of formula I includes pharmaceutically acceptable salts. Suitable salts include inorganic acid (e.g. hydrogen halide), and organic acid (e.g. acetic, methanesulfonic or trifluoroacetic acid), addition salts.
The alkyl part of alkoxy groups which R2 may represent, may, when there is a sufficient number of carbon atoms, be linear or branched, be saturated or unsaturated, be cyclic, acyclic or part cyclic/acyclic, and/or be optionally interrupted by an O atom.
Halo groups which R1 and R2 may represent, and with which R2 may be substituted, include fluoro, chloro, bromo and iodo.
Abbreviations are listed at the end of this specification.
Compounds of the invention that may be mentioned include those in which Y represents S(O)2.
Preferred compounds of the invention include those in which:
R1 represents chloro;
R2 represents H, halo or C1-2 alkoxy (which latter group is optionally substituted by one or more halo (e.g. fluoro) groups).
More preferred compounds of the invention include those in which:
R1 represents chloro; and
R2 represents H, chloro, OCHF2, OCF3 or, especially, OCH3.
Preferred compounds of formula I include the compounds of the Examples described hereinafter, particularly the compound of Example 1.
According to the invention there is also provided a process for the preparation of compounds of formula I which comprises:
(i) the coupling of a compound of formula III, 
xe2x80x83wherein Y, R1 and R2 are as hereinbefore defined, with 4-amidinobenzyl-2-azetidinecarboxamide (see, for example, international patent application WO 97/02284), for example in the presence of a coupling agent (e.g. EDC, DCC, HBTU, HATU, TBTU, PyBOP or oxalyl chloride in DMF), an appropriate base (e.g. pyridine, 2,4,6-collidine, DMAP, TEA or DIPEA) and a suitable organic solvent (e.g. dichloromethane, acetonitrile or DMF);
(ii) the coupling of a compound of formula IV, 
xe2x80x83wherein Y, R1 and R2 are as hereinbefore defined, with para-amidino-benzylamine, for example in the presence of a coupling agent (e.g. oxalyl chloride in DMF, EDC, DCC, HBTU, HATU, PyBOP or TBTU), an appropriate base (e.g. pyridine, DMAP, TEA, 2,4,6-collidine or DIPEA) and a suitable organic solvent (e.g. dichloromethane, acetonitrile or DMF); or
(iii) complete oxidation (for compounds of formula I in which Y is S(O)2), or partial oxidation (for compounds of formula I in which Y is S(O)), of a corresponding compound of formula V, 
xe2x80x83wherein R1 and R2 are as hereinbefore defined, for example in the presence of an appropriate amount of a suitable oxidising agent (e.g. mCPBA, hydrogen peroxide or potassium peroxymonosulfate) and an appropriate organic solvent (e.g. CH2Cl2, methanol, water or mixtures thereof), and optionally in the presence of a suitable protic acid (e.g. acetic acid).
The skilled person will appreciate that, in the case of partial oxidation, a mixture of stereoisomers may be obtained, which may be separated by techniques known to those skilled in the art (e.g. by column chromatography or chiral chromatography).
Compounds of formula III may be prepared by complete or partial oxidation of a compound of formula VI, 
wherein R1 and R2 are as hereinbefore defined, for example under conditions such as those described hereinbefore for the synthesis of compounds of formula I (process step (iii)).
Compounds of formula IV may be prepared by the coupling of a compound of formula III, as hereinbefore defined, with azetidine-2-carboxylic acid, for example under conditions such as those described hereinbefore for synthesis of compounds of formula I (see, for example, process steps (i) and (ii)).
Compounds of formula IV may alternatively be prepared by complete or partial oxidation of a compound of formula VII, 
wherein R1 and R2 are as hereinbefore defined, for example under conditions such as those described hereinbefore for the synthesis of compounds of formula I (process step (iii)).
Compounds of formula V may be prepared in accordance with peptide coupling techniques, for example in analogous fashion to the methods described hereinbefore for compounds of formula I (see, for example, process steps (i) and (ii)). If desired, compounds of formula VII may also be prepared in this way.
Compounds of formula VI are available using known and/or standard techniques.
For example, compounds of formula VI may be prepared by reaction of a compound of formula VIII, 
wherein R1 and R2 are as hereinbefore defined, with:
(a) a compound of formula IX,
Rxe2x80x3 CNxe2x80x83xe2x80x83IX
wherein Rxe2x80x3 represents H or (CH3)3Si, for example at room, or elevated, temperature (e.g. below 100xc2x0 C.) in the presence of a suitable organic solvent (e.g. chloroform or dichloromethane) and, if necessary, in the presence of a suitable base (e.g. TEA) and/or a suitable catalyst system (e.g. benzylammonium chloride or zinc iodide), followed by hydrolysis in the presence of an acid (e.g. HCl or H2SO4), for example at 20xc2x0 C. (e.g. according, or analogously, to the method described by C. F. Bigge et al. in J. Med. Chem. (1993) 36, 1977), and then followed by hydrolysis under alkaline conditions (e.g. in the presence of water and either lithium or potassium hydroxide) to give the free acid;
(b) NaCN or KCN, for example in the presence of NaHSO3 and water, followed by hydrolysis; or
(c) chloroform, for example at elevated temperature (e.g. above room temperature but below 100xc2x0 C.) in the presence of a suitable base (e.g. sodium hydroxide) and, if necessary, in the presence of a suitable catalyst system (e.g. benzylammonium chloride), followed by hydrolysis.
The enantiomeric forms of compounds of formula VI (i.e. those compounds having different configurations of substituents about the C-atom that is in the xcex1-position relative to the CO2H group) may be separated by techniques known to those skilled in the art (e.g. by chromatography, using a chiral chromatographic medium).
Compounds of formula VI may alternatively be prepared by way of a Sharpless stereoselective dihydroxylation of a compound of formula X, 
wherein R1 and R2 are as hereinbefore defined, under conditions known to those skilled in the art (e.g. at low temperature (e.g. 0xc2x0 C.), using, for example, the commercial reagent AD-mix-(trademark) in the presence of suitable solvent (e.g. t-butanol), followed by oxidation of the resultant intermediate (e.g. at elevated temperature (e.g. 75xc2x0 C.) in the presence of a stream of air and Pt/C (5%) in acetone/water).
Compounds of formula VIII are available using known and/or standard techniques. For example, compounds of formula VIII may be prepared by cyclisation of a compound of formula XI, 
wherein L1 represents a leaving group such as OH, C1-4 alkoxy, C1-4 acyloxy (optionally substituted by one or more halo atoms) or halo and R1 and R2 are as hereinbefore defined, for example in the presence of a suitable catalyst (e.g. trifluoroacetic anhydride, a protic acid such as H2SO4 or a Lewis acid such as BF3) and optionally in the presence of an appropriate solvent (e.g. CH2Cl2).
Compounds of formula XI may be prepared in accordance with known techniques. For example, compounds of formula XI may be prepared by reaction of a compound of formula XII, 
wherein R1 and R2 are as hereinbefore defined, with a compound of formula XIII, 
wherein L2 represents a leaving group such as halo and Rx represents C1-6 alkyl, for example at between room and reflux temperature in the presence of an appropriate base (e.g. triethylamine or Cs2CO3) and a suitable solvent (e.g. ethyl acetate or acetone), followed by conversion of the ORX group into an L1 group under conditions that are well known to those skilled in the art (e.g. for compounds of formula XI in which L1 represents OH, by hydrolysis under alkaline conditions).
Compounds of formulae IX, X, XII and XIII, and derivatives thereof, are either commercially available, are known in the literature, or may be obtained either by analogy with the processes described herein, or by conventional synthetic procedures, in accordance with standard techniques, from readily available starting materials using appropriate reagents and reaction conditions (e.g. as described hereinafter).
The compounds of formula I may be isolated from their reaction mixtures using conventional techniques.
Substituents on the aromatic ring in compounds of formulae I, III, IV, V, VI, VII, VIII, X, XI and XII may be introduced and/or interconverted using techniques well known to those skilled in the art. For example, halo may be introduced, both into the aromatic ring and into the alkyl part of alkoxy groups that R2 may represent, by reaction with suitable halogenating agents, for example as described hereinafter.
In accordance with the present invention, pharmaceutically acceptable derivatives of compounds of formula I also include xe2x80x9cprotectedxe2x80x9d derivatives of compounds of formula I and/or compounds that act as prodrugs of compounds of formula I.
In this respect, according to a further aspect of the invention there is provided derivatives of compounds of formula I as defined herein which are compounds of formula IA, 
wherein
Y, R1 and R2 are as hereinbefore defined;
D1 and D2 independently represent H, xe2x80x94OR7 or R8, or D1, D2 and R3, together with the amidine group to which they are attached, form a cyclic group of formula IIa, IIb, IIc, IId or IIe, 
xe2x80x83wherein wavy lines indicate the points of attachment to the benzene ring;
R3 represents H, or R3, D1, and D2 together with the amidine group to which they are attached, form a cyclic group of formula IIa, IIb, IIc, IId or IIe;
R4 and R5 independently represent H or C1-4 alkyl;
R6 represents H, C1-6 alkyl (which latter group is optionally substituted by one or more halo groups) or C(O)OR12;
R7 represents H, C6-10 aryl, C1-10 alkyl (which latter group is optionally substituted by one or more halo groups), C1-3 alkylphenyl, xe2x80x94C(R9a)(R9b)R10, xe2x80x94C(O)R11a, xe2x80x94C(O)O12, xe2x80x94C(O)N(R13)R14 or xe2x80x94(CH2)n(O)mR15;
R8 represents xe2x80x94C(R9a)(R9b)R10, xe2x80x94C(O)R11b or xe2x80x94C(O)OR12;
R9a and R9b independently represent, at each occurrence, H or C1-6 alkyl;
R10 represents, at each occurrence, xe2x80x94OC(O)R16a, xe2x80x94OC(O)OR17, xe2x80x94N(R18a)C(O)OR17 or xe2x80x94OC(O)N(R18b)R17;
R11a and R11b independently represent, at each occurrence, C6-10 aryl, C1-3 alkylphenyl (which latter two groups are optionally substituted by one or more substituents selected from C1-6 alkyl and halo), xe2x80x94[C(R19a)(R19b)]pOC(O)R16b, or R11a represents C1-17 alkyl (optionally substituted by C1-6 alkoxy, C1-6 acyloxy, amino or halo) or R11b represents C1-6 alkyl;
R12 represents, at each occurrence, C1-17 alkyl (optionally substituted by one or more substituents selected from C1-6 alkoxy, C1-6 acyloxy, xe2x80x94Si(R20a)(R20b)(R21c) and halo), C6-10 aryl, C1-3 alkylphenyl (which latter two groups are optionally substituted by C1-6 alkyl, C1-6 alkoxy, cyano or halo), xe2x80x94[C(R19a)(R19b)]qOC(O)R16b or xe2x80x94CH2R21;
R13 represents H or C1-7 alkyl, or together with R14 represents C4-5 alkylene;
R14 represents C6-10 aryl or C1-10 alkyl (which latter group is optionally substituted by one or more substituents selected from OH, halo, CO2H, C1-6 alkoxy, C1-6 acyloxy and C6-10 aryl), or together with R13 represents C4-5 alkylene;
R15 represents C1-7 alkyl optionally substituted by one or more xe2x80x94OC(O)C(H)(R22)N(G)(Ga) groups;
R16a, R16b and R17 independently represent, at each occurrence, C6-10 aryl or C1-17 alkyl (which latter group is optionally substituted by one or more substituents selected from xe2x80x94OH, halo, xe2x80x94CO2H, C1-6 alkoxy, C1-6 acyloxy and C6-10 aryl), or R16b represents C1-6 alkoxy (optionally substituted by one or more substituents selected from C1-6 alkyl and halo);
R11a and R11b independently represent H or C1-4 alkyl;
R19a and R19b independently represent, at each occurrence, H or C1-6 alkyl;
R20a to R20c independently represent, at each occurrence, C1-6 alkyl or phenyl;
R21 represents the structural fragment IIf 
R22 represents C3-4 alkyl;
G and Ga independently represent H, an amino protective group, or G and
Ga together represent an amino protective group;
m represents 0 or 1;
n represents 1, 2 or 3;
p represents 3 or 4;
q represents 2 or 3;
or a pharmaceutically acceptable salt thereof,
provided that:
(a) D1 and D2 do not both represent H; and
(b) when one of D1 and D2 represents xe2x80x94OR7, then the other represents H.
Suitable xe2x80x9cpharmaceutically acceptable saltsxe2x80x9d of compounds of formula IA include inorganic acid (e.g. hydrogen halide), and organic acid (e.g. acetic, methanesulfonic or trifluoroacetic acid), addition salts.
The compounds of the invention may exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of the invention. Particular tautomeric forms that may be mentioned include those connected with the position of the double bond in the amidine functionality, and the position of the substituent D1 or D2, in compounds of formula IA.
For the avoidance of doubt, in compounds of formula IA, substituents D1 and D2 are completely independent of each other. For example, D1 and D2 may both be represented by R8 in which, in both cases, R8 represents xe2x80x94C(O)R11b in which R11b represents, in both cases, C1-6 alkyl. However, in such instances the C1-6 alkyl group may be the same of different.
The compounds of formulae I and IA also contain at least two asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism. All diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques. Alternatively the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation, or by derivatisation, for example with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means (e.g. HPLC, chromatography over silica). All stereoisomers are included within the scope of the invention.
As used herein, the term xe2x80x9carylxe2x80x9d includes phenyl, naphthyl (e.g. 2-naphthyl) and the like. Unless otherwise indicated, aryl groups are optionally substituted by one or more substituents selected form C1-6 alkyl and halo.
Alkyl and alkylene groups, as well as alkyl parts of alkoxy, alkylphenyl and acyloxy groups, in compounds of formula IA may, when there is a sufficient number of carbon atoms, be linear or branched, be saturated or unsaturated, be cyclic, acyclic or part cyclic/acyclic, and/or be optionally interrupted by an O atom. The skilled person will appreciate that, when alkyl groups in compounds of formula IA are cyclic and interrupted by oxygen, they may then represent oxygen-containing heterocycles such as tetrahydrofuranyl or (where appropriate) tetrahydropyranyl.
Halo groups in compounds of formula IA include fluoro, chloro, bromo and iodo.
As used herein, the term xe2x80x9camino protective groupxe2x80x9d includes groups mentioned in xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d, 2nd edition, T W Greene and P G M Wutz, Wiley-Interscience (1991), in particular those indexed at the start of the chapter entitled xe2x80x9cProtection for the Amino Groupxe2x80x9d (see pages 309 to 315) of that reference, the disclosure in which document is hereby incorporated by reference.
Specific examples of amino protective groups thus include:
(a) carbamate groups (e.g. methyl, cyclopropylmethyl, 1-methyl-1-cyclopropylmethyl, diisopropylmethyl, 9-fluorenylmethyl, 9-(2-sulfo)fluorenylmethyl, 2-furanylmethyl, 2,2,2-trichloroethyl, 2-haloethyl, 2-trimethylsilylethyl, 2-methylthioethyl, 2-methyl-sulfonylethyl, 2(p-toluenesulfonyl)ethyl, 2-phosphonioethyl, 1,1-dimethylpropynyl, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)-propyl, 1,1-dimethyl-3-(N,N-diethylamino)propyl, 1-methyl-1-(1-adamantyl)ethyl, 1-methyl-1-phenylethyl, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl, 1-methyl-1-(4-biphenylyl)ethyl, 1-methyl-1-(p-phenylazophenyl)ethyl, 1,1-dimethyl-2-haloethyl, 1,1-dimethyl-2,2,2-trichloroethyl, 1,1-dimethyl-2-cyanoethyl, isobutyl, t-butyl, t-amyl, cyclobutyl, 1-methylcyclobutyl, cyclopentyl, cyclohexyl, 1-methylcyclohexyl, 1-adamantyl, isobornyl, vinyl, allyl, cinnamyl, phenyl, 2,4,6-tri-t-butylphenyl, m-nitrophenyl, S-phenyl, 8-quinolinyl, N-hydroxypiperidinyl, 4-(1,4-dimethylpiperidinyl), 4,5-diphenyl-3-oxazolin-2-one, benzyl, 2,4,6-trimethylbenzyl, p-methoxybenzyl, 3,5-dimethoxybenzyl, p-decyloxybenzyl, p-nitrobenzyl, o-nitrobenzyl, 3,4-dimethoxy-6-nitrobenzyl, p-bromobenzyl, chlorobenzyl, 2,4-dichloro-benzyl, p-cyanobenzyl, o-(N,N-dimethylcarboxamidobenzyl)benzyl, m-chloro-p-acyloxybenzyl, p-(dihydroxyboryl)benzyl, p-(phenylazo)benzyl, p-(pxe2x80x2-methoxyphenylazo)benzyl, 5-benzisoxazolylmethyl, 9-anthrylmethyl, diphenylmethyl, phenyl(o-nitrophenyl)methyl, di(2-pyridyl)methyl, 1-methyl-1-(4-pyridyl)-ethyl, isonicotinyl or S-benzyl carbamate groups);
(b) amide groups (e.g. N-formyl, N-acetyl, N-chloroacetyl, N-dichloroacetyl, N-trichloroacetyl, N-trifluoroacetyl, N-o-nitrophenylacetyl, N-o-nitrophenoxyacetyl, N-acetoacetyl, N-acetylpyridinium, N-3-phenylpropionyl, N-3-(p-hydroxyphenyl)propionyl, N-3-(o-nitrophenyl)propionyl, N-2-methyl-2-(o-nitrophenoxy)propionyl, N-2-methyl-2-(o-phenylazophenoxy)propionyl, N-4-chlorobutyryl, N-isobutyryl, N-o-nitrocinnamoyl, N-picolinoyl, N-(Nxe2x80x2-acetylmethionyl), N-(Nxe2x80x2-benzoylphenylalanyl), N-benzoyl, N-p-phenyl-benzoyl, N-p-methoxybenzoyl, N-o-nitrobenzoyl or N-o-(benzoyloxymethyl)benzoyl amide groups);
(c) alkyl groups (e.g. N-allyl, N-phenacyl, N-3-acetoxypropyl, N-(4-nitro-1-cyclohexyl-2-oxo-pyrrolin-3-yl), N-methoxymethyl, N-chloroethoxymethyl, N-benzyloxymethyl, N-pivaloyloxymethyl, N-2-tetrahydropyranyl, N-2,4-dinitrophenyl, N-benzyl, N-3,4-dimethoxybenzyl, N-o-nitrobenzyl, N-di(p-methoxyphenyl)methyl, N-triphenylmethyl, N-(p-methoxyphenyl)diphenylmethyl, N-diphenyl-4-pyridylmethyl, N-2-picolyl Nxe2x80x2-oxide or N-dibenzosuberyl groups);
(d) phosphinyl and phosphoryl groups (e.g. N-diphenylphosphinyl, N-dimethylthiophosphinyl, N-diphenylthiophosphinyl, N-diethylphosphoryl, N-dibenzylphosphoryl or N-phenylphosphoryl groups);
(e) sulfenyl groups (e.g. N-benzenesulfenyl, N-o-nitrobenzenesulfenyl, N-2,4-dinitrobenzenesulfenyl, N-pentachlorobenzenesulfenyl, N-2-nitro-4-methoxybenzenesulfenyl or N-triphenylmethylsulfenyl groups);
(f) sulfonyl groups (e.g. N-benzenesulfonyl, N-p-methoxybenzenesulfonyl, N-2,4,6-trimethylbenzenesulfonyl, N-toluenesulfonyl, N-benzylsulfonyl, N-p-methylbenzylsulfonyl, N-trifluoromethylsulfonyl or N-phenacylsulfonyl); and
(g) the N-trimethylsilyl group.
Compounds of formulae I and IA in which the fragment 
is in the S-configuration are preferred. Compounds of formulae I and IA in which the fragment 
is in the R-configuration are preferred.
The wavy lines on the bonds in the above two fragments signify the bond positions of the fragments.
Compounds of formula IA may be prepared directly from compounds of formula I, in an analogous manner to compounds of formula I, or using procedures appropriately adapted from those used to prepare compounds of formula I. In this respect, processes that may be used to prepare compounds of formula IA include:
(1) for compounds of formula IA in which one of D1 and D2 represents xe2x80x94OR7 (in which R7 represents H, C6-10 aryl, C1-10 alkyl (which latter group is optionally substituted by one or more halo groups) or C1-3 alkylphenyl), reaction of a compound of formula XIV, 
wherein Y, R1 and R2 are as hereinbefore defined, with a compound of formula XV,
H2NORaxe2x80x83xe2x80x83XV
wherein Ra represents H, C6-10 aryl, C1-10 alkyl (which latter group is optionally substituted by one or more halo groups) or C1-3 alkylphenyl, for example at between 40 and 60xc2x0 C., in the presence of a suitable base (e.g. TEA) and an appropriate organic solvent (e.g. THF, CH3CN, DMF or DMSO), optionally by pre-treating the compound of formula XIV with gaseous HCl, in the presence of a lower alkyl (e.g. C1-6 alkyl) alcohol (e.g. ethanol) at, for example, 0xc2x0 C., to form a compound of formula XVI, 
wherein Rb represents lower (e.g. C1-6) alkyl, such as ethyl, and Y, R1 and R2 are as hereinbefore defined, which compound may be isolated if desired;
(2) for compounds of formula IA in which one of D1 and D2 represents xe2x80x94OR7 (in which R7 represents H, C6-10 aryl, C1-10 alkyl (which latter group is optionally substituted by one or more halo groups) or C1-3 alkylphenyl), reaction of a corresponding compound of formula IA, in which one of D1 and D2 represents xe2x80x94C(O)OR12 (e.g. xe2x80x94C(O)Oxe2x80x94CH2CH2xe2x80x94Si(CH3)3) and the other represents H, with a compound of formula XV, as hereinbefore defined, for example at between room and reflux temperature in the presence of an appropriate organic solvent (e.g. THF, CH3CN, DMF or DMSO), followed by removal of the xe2x80x94C(O)OR12 group under conditions known to those skilled in the art;
(3) for compounds of formula IA in which D1 or D2 represents R8, reaction of a corresponding compound of formula I or a corresponding compound of formula IA in which D1 or D2 (as appropriate) represents H with a compound of formula XVII,
L3xe2x80x94R8xe2x80x83xe2x80x83XVII
wherein L3 represents a leaving group, such as halo or p-nitrophenoxy, and R8 is as hereinbefore defined, for example at between 0xc2x0 C. and room temperature in the presence of a suitable base (e.g. NaOH) and an appropriate organic solvent (e.g. THF) and/or water;
(4) for compounds of formula IA in which one of D1 and D2 represents xe2x80x94OR7 (wherein R7 does not represent H), reaction of a corresponding compound of formula IA in which one of D1 and D2 represents xe2x80x94OH with a compound of formula XVIII,
L3xe2x80x94R7axe2x80x83xe2x80x83XVIII
wherein R7a represents R7 as hereinbefore defined, except that it does not represent H, and L3 is as hereinbefore defined, for example at between 0xc2x0 C. and reflux temperature, optionally in the presence of an appropriate solvent (e.g. DCM, THF, MeCN or DMF) and a suitable base (e.g. Et3N or pyridine); or
(5) for compounds of formula IA in which one of D1 and D2 represents H and the other represents xe2x80x94C(R9a)(R9b)R10, wherein R10 represents xe2x80x94OC(O)R16a, xe2x80x94OC(O)OR17 or xe2x80x94OC(O)N(R18b)R17, reaction of a corresponding compound of formula XIX, 
wherein Y, R1, R2, R9a and R9b are as hereinbefore defined with a compound of formula XX,
L3xe2x80x94C(O)Rcxe2x80x83xe2x80x83XX
wherein Rc represents R16a, xe2x80x94OR17 or xe2x80x94N(R18b)R17, and L3, R6, R17 and R18b are as hereinbefore defined, for example under conditions described hereinbefore (e.g. as in step (4) above).
Compounds of formula XIV may be prepared in an analogous fashion to compounds of formula I, for example by the coupling of p-cyano-benzylamine with a compound of formula IV, as hereinbefore defined, under conditions such as those described hereinbefore for the synthesis of compounds of formula I (see, for example, process steps (i) and (ii)).
Compounds of formula XIX may be prepared by reaction of a corresponding compound of formula I with an excess of a compound of formula XXI,
R9aC(O)R9bxe2x80x83xe2x80x83XXI
wherein R9a and R9b are as hereinbefore defined, for example under conditions known to those skilled in the art.
Compounds of formula IA in which D1, D2 and R3, together with the amidine group to which they are attached, represent a group IIa may be prepared by reaction of a corresponding compound of formula IA in which D1 represents OH with a compound of formula XXII,
HalC(O)CH(R4)Halxe2x80x83xe2x80x83XXII
wherein Hal represents halo (e.g. chloro) and R4 is as hereinbefore defined, for example at room temperature in the presence of a suitable base (e.g. triethylamine) and an appropriate organic solvent (e.g. dichloromethane), followed by cyclisation of the resultant intermediate, for example by refluxing in the presence of an appropriate base (e.g. sodium hydride) in a suitable organic solvent (e.g. THF).
Compounds of formula IA in which D1, D2 and R3, together with the amidine group to which they are attached, represent a group IIb may be prepared by reaction of a corresponding compound of formula XXIII, 
wherein Y, R1 and R2 are as hereinbefore defined with a compound of formula XXIV,
H2NCH(R4)C(O)ORbxe2x80x83xe2x80x83XXIV
wherein Rb and R4 are as hereinbefore defined, for example at room temperature in the presence of a suitable base (e.g. triethylamine) and an appropriate organic solvent (e.g. ethanol).
Compounds of formula IA in which D1, D2 and R3, together with the amidine group to which they are attached, represent a group IIc may be prepared by reaction of a corresponding compound of formula IA in which D1 represents OH with a compound of formula XXV,
R4CHOxe2x80x83xe2x80x83XXV
wherein R4 is as hereinbefore defined, for example at room temperature, followed by oxidation of the resultant intermediate under conditions known to those skilled in the art.
Compounds of formula IA in which D1, D2 and R3, together with the amidine group to which they are attached, represent a group IId may be prepared by cyclisation of a compound of formula XXVI, 
wherein Hal is as hereinbefore defined (especially iodo) and Y, R1, R2 R4, R5 and R6 are as hereinbefore defined, for example at room temperature in the presence of a suitable base (e.g. DIPEA) and an appropriate solvent (e.g. dichloromethane).
Compounds of formula XXVI may be prepared by reaction of a compound of formula XXVII, 
wherein Y, R1, R2 and R6 are as hereinbefore defined with a compound of formula XXVIII,
Halxe2x80x94C(O)OC(R4)(R5)Halxe2x80x83xe2x80x83XXVIII
wherein Hal, R4 and R1 are as hereinbefore defined for example at room temperature in the presence of a suitable base (e.g. triethylamine) and an appropriate organic solvent (e.g. dichloromethane). It is preferred that, in compounds of formula XXVIII, Hal represents chloro. In such cases, the intermediate of formula XXVI that is formed comprises a chloro group, which, before cyclisation, is preferably converted to an iodo group, using techniques well know to those skilled in the art (e.g. reaction with NaI).
Compounds of formula IA in which D1, D2 and R3, together with the amidine group to which they are attached, represent a group IIe may be prepared by reaction of a corresponding compound of formula XXIX, 
wherein Y, R1, R2 and R6 are as hereinbefore defined with formaldehyde in water, for example at reflux temperature.
Compounds of formulae XV, XVII, XVIII, XX, XXI, XXII, XXIII, XXIV, XXV, XXVII, XXVIII and XXIX and derivatives thereof, are either commercially available, are known in the literature, or may be obtained either by analogy with the processes described herein, or by conventional synthetic procedures, in accordance with standard techniques, from readily available starting materials using appropriate reagents and reaction conditions (e.g. as described hereinafter).
Compounds of formula IA may be isolated from their reaction mixtures using conventional techniques.
Substituents on the aromatic and/or non-aromatic, carbocyclic and heterocyclic ring(s) in compounds of formulae IA, XIV, XV, XVI, XVII, XVIII, XIX, XX, XXI, XXIII, XXVI, XXVIII and XXIX may be introduced and/or interconverted using techniques well known to those skilled in the art. For example, hydroxy may be alkylated to give alkoxy or acylated to give acyloxy, and alkoxy and acyloxy may be hydrolysed to give hydroxy.
It will be appreciated by those skilled in the art that in the processes described above (in relation to compounds of formulae I and IA) the functional groups of intermediate compounds may need to be protected by protecting groups.
Functional groups that it is desirable to protect include hydroxy, amino, aldehyde, ketone, 2-hydroxycarboxylic acid and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl groups (e.g. t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl) and tetrahydropyranyl. Suitable protecting groups for carboxylic acid include C1-6 alkyl or benzyl esters. Suitable protecting groups for amino and amidino include t-butyloxycarbonyl, benzyloxycarbonyl or 2-trimethylsilylethoxycarbonyl (Teoc). Amidino nitrogens may also be protected by hydroxy or alkoxy groups, and may be either mono- or diprotected. Aldehydes and ketones may be protected as acetals and ketals, respectively, by reacting with e.g. ethylene glycol. 2-Hydroxy carboxylic acids may be protected by condensing with e.g. acetone.
The protection and deprotection of functional groups may take place before or after coupling, or before or after any other reaction in the abovementioned schemes.
Protecting groups may be removed in accordance with techniques that are well known to those skilled in the art and as described hereinafter. Persons skilled in the art will appreciate that, in order to obtain compounds of formula I, or formula IA, in an alternative, and, on some occasions, more convenient, manner, the individual process steps mentioned hereinbefore may be performed in a different order, and/or the individual reactions may be performed at a different stage in the overall route (i.e. substituents may be added to and/or chemical transformations performed upon, different intermediates to those mentioned hereinbefore in conjunction with a particular reaction). This may negate, or render necessary, the need for protecting groups.
For example, this is particularly true in respect of the synthesis of compounds of formula IA. In this case, D1 or D2 groups which do not represent H may be introduced at an earlier stage in the overall synthesis using the process steps described hereinbefore (see, for example, steps (1) to (5) above). Further, in the synthesis of compounds of formula I, the xe2x80x94OH group that is in the xcex1-position relative to the carboxylic acid in compounds of formulae III and IV may need to be protected prior to the coupling steps described above.
Accordingly, the order and type of chemistry involved will dictate the need, and type, of protecting groups as well as the sequence for accomplishing the synthesis.
The use of protecting groups is fully described in xe2x80x9cProtective Groups in Organic Chemistryxe2x80x9d, edited by J W F McOmie, Plenum Press (1973), and xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d, 3rd edition, T W Greene and P G M Wutz, Wiley-Interscience (1999).
Protected derivatives of compounds of formulae I and IA may be converted chemically to compounds of formulae I and IA using standard deprotection techniques (e.g. hydrogenation).
Compounds of the invention may possess pharmacological activity as such. Compounds of the invention that may possess such activity include, but are not limited to, compounds of formula I.
However, other compounds of the invention (including compounds of formula IA) may not possess such activity, but may be administered parenterally or orally, and thereafter metabolised in the body to form compounds that are pharmacologically active (including, but not limited to, corresponding compounds of formula I). Such compounds (which also includes compounds that may possess some pharmacological activity, but that activity is appreciably lower than that of the xe2x80x9cactivexe2x80x9d compounds to which they are metabolised), may therefore be described as xe2x80x9cprodrugsxe2x80x9d of the active compounds.
Thus, the compounds of the invention are useful because they possess pharmacological activity, and/or are metabolised in the body following oral or parenteral administration to form compounds which possess pharmacological activity. The compounds of the invention are therefore indicated as pharmaceuticals.
According to a further aspect of the invention there is thus provided the compounds of the invention for use as pharmaceuticals.
In particular, compounds of the invention are potent inhibitors of thrombin either as such and/or (e.g. in the case of prodrugs), are metabolised following administration to form potent inhibitors of thrombin, for example as demonstrated in the tests described below.
By xe2x80x9cprodrug of a thrombin inhibitorxe2x80x9d, we include compounds that form a thrombin inhibitor, in an experimentally-detectable amount, and within a predetermined time (e.g. about 1 hour), following oral or parenteral administration.
The compounds of the invention are thus expected to be useful in those conditions where inhibition of thrombin is required.
The compounds of the invention are thus indicated in the treatment and/or prophylaxis of thrombosis and hypercoagulability in blood and tissues of animals including man.
It is known that hypercoagulability may lead to thrombo-embolic diseases. Conditions associated with hypercoagulability and thrombo-embolic diseases which may be mentioned include inherited or acquired activated protein C resistance, such as the factor V-mutation (factor V Leiden), and inherited or acquired deficiencies in antithrombin III, protein C, protein S or heparin cofactor II. Other conditions known to be associated with hypercoagulability and thrombo-embolic disease include circulating antiphospholipid antibodies (Lupus anticoagulant), homocysteinemi, heparin induced thrombocytopenia and defects in fibrinolysis. The compounds of the invention are thus indicated both in the therapeutic and/or prophylactic treatment of these conditions.
The compounds of the invention are further indicated in the treatment of conditions where there is an undesirable excess of thrombin without signs of hypercoagulability, for example in neurodegenerative diseases such as Alzheimer""s disease.
Particular disease states which may be mentioned include the therapeutic and/or prophylactic treatment of venous thrombosis and pulmonary embolism, arterial thrombosis (e.g. in myocardial infarction, unstable angina, thrombosis-based stroke and peripheral arterial thrombosis) and systemic embolism usually from the atrium during arterial fibrillation or from the left ventricle after transmural myocardial infarction or caused by congestive heart failure.
Moreover, the compounds of the invention are expected to have utility in prophylaxis of re-occlusion (i.e. thrombosis) after thrombolysis, percutaneous trans-luminal angioplasty (PTA) and coronary bypass operations; the prevention of re-thrombosis after microsurgery and vascular surgery in general.
Further indications include the therapeutic and/or prophylactic treatment of disseminated intravascular coagulation caused by bacteria, multiple trauma, intoxication or any other mechanism; anticoagulant treatment when blood is in contact with foreign surfaces in the body such as vascular grafts, vascular stents, vascular catheters, mechanical and biological prosthetic valves or any other medical device; and anticoagulant treatment when blood is in contact with medical devices outside the body such as during cardiovascular surgery using a heart-lung machine or in haemodialysis.
In addition to its effects on the coagulation process, thrombin is known to activate a large number of cells (such as neutrophils, fibroblasts, endothelial cells and smooth muscle cells). Therefore, the compounds of the invention may also be useful for the therapeutic and/or prophylactic treatment of idiopathic and adult respiratory distress syndrome, pulmonary fibrosis following treatment with radiation or chemotherapy, septic shock, septicemia, inflammatory responses, which include, but are not limited to, edema, acute or chronic atherosclerosis such as coronary arterial disease, cerebral arterial disease, peripheral arterial disease, reperfusion damage, and restenosis after percutaneous trans-luminal angioplasty (PTA).
Compounds of the invention that inhibit trypsin and/or thrombin may also be useful in the treatment of pancreatitis.
According to a further aspect of the present invention, there is provided a method of treatment of a condition where inhibition of thrombin is required which method comprises administration of a therapeutically effective amount of a compound of the invention to a person suffering from, or susceptible to such a condition.
The compounds of the invention will normally be administered orally, intravenously, subcutaneously, buccally, rectally, dermally, nasally, tracheally, bronchially, by any other parenteral route or via inhalation, in the form of pharmaceutical preparations comprising active compound either as a free base, or a pharmaceutically acceptable non-toxic organic or inorganic acid addition salt, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated and the route of administration, the compositions may be administered at varying doses.
The compounds of the invention may also be combined and/or co-administered with any antithrombotic agent with a different mechanism of action, such as the antiplatelet agents acetylsalicylic acid, ticlopidine, clopidogrel, thromboxane receptor and/or synthetase inhibitors, fibrinogen receptor antagonists, prostacyclin mimetics and phosphodiesterase inhibitors, ADP-receptor (P2T) antagonists and inhibitors of carboxypeptidase U (CPU).
The compounds of the invention may further be combined and/or co-administered with thrombolytics such as tissue plasminogen activator (natural, recombinant or modified), streptokinase, urokinase, prourokinase, anisoylated plasminogen-streptokinase activator complex (APSAC), animal salivary gland plasminogen activators, and the like, in the treatment of thrombotic diseases, in particular myocardial infarction.
According to a further aspect of the invention there is thus provided a pharmaceutical formulation including a compound of the invention, in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.
Suitable daily doses of the compounds of the invention in therapeutic treatment of humans are about 0.001-100 mg/kg body weight at peroral administration and 0.001-50 mg/kg body weight at parenteral administration.
The compounds of the invention have the advantage that they may, or may be metabolised to compounds that may, be more efficacious, be less toxic, be longer acting, have a broader range of activity, be more potent, produce fewer side effects, be more easily absorbed, or have a better pharmacokinetic profile, than, or have other useful pharmacological, physical, or chemical, properties over, compounds known in the prior art.
The following test procedures may be employed.
Test A
Determination of Thrombin Clotting Time (TT)
The inhibitor solution (25 L) is incubated with plasma (25 L) for three minutes. Human thrombin (T 6769; Sigma Chem. Co or Hematologic Technologies) in buffer solution, pH 7.4 (25 L, 4.0 NIH units/mL), is then added and the clotting time measured in an automatic device (KC 10; Amelung).
The thrombin clotting time (TT) is expressed as absolute values (seconds) as well as the ratio of TT without inhibitor (TT0) to TT with inhibitor (TTi). The latter ratios (range 1-0) are plotted against the concentration of inhibitor (log transformed) and fitted to sigmoidal dose-response curves according to the equation
y=a/[1+(x/IC50)s]
where: a=maximum range, i.e. 1; s=slope of the dose-response curve; and IC50=the concentration of inhibitor that doubles the clotting time. The calculations are processed on a PC using the software program GraFit Version 3, setting equation equal to: Start at 0, define end=1 (Erithacus Software, Robin Leatherbarrow, Imperial College of Science, London, UK).
Test B
Determination of Thrombin Inhibition With a Chromogenic, Robotic Assay
The thrombin inhibitor potency is measured with a chromogenic substrate method, in a Plato 3300 robotic microplate processor (Rosys AG, CH-8634 Hombrechtikon, Switzerland), using 96-well, half volume microtitre plates (Costar, Cambridge, Mass., USA; Cat No 3690). Stock solutions of test substance in DMSO (72 L), 0.1xe2x88x921 mmol/L, are diluted serially 1:3 (24+48 L) with DMSO to obtain ten different concentrations, which are analysed as samples in the assay. 2 L of test sample is diluted with 124 L assay buffer, 12 L of chromogenic substrate solution (S-2366, Chromogenix, Mxc3x6lndal, Sweden) in assay buffer and finally 12 L of -thrombin solution (Human (xcex1-thrombin, Sigma Chemical Co. or Hematologic Technologies) in assay buffer, are added, and the samples mixed. The final assay concentrations are: test substance 0.00068xe2x88x9213.3 mol/L, S-2366 0.30 mmol/L, -thrombin 0.020 NIHU/mL. The linear absorbance increment during 40 minutes incubation at 37xc2x0 C. is used for calculation of percentage inhibition for the test samples, as compared to blanks without inhibitor. The IC50-robotic value, corresponding to the inhibitor concentration which causes 50% inhibition of the thrombin activity, is calculated from a log concentration vs. % inhibition curve.
Test C
Determination of the Inhibition Constant Ki for Human Thrombin
Ki-determinations are made using a chromogenic substrate method, performed at 37xc2x0 C. on a Cobas Bio centrifugal analyser (Roche, Basel, Switzerland). Residual enzyme activity after incubation of human -thrombin with various concentrations of test compound is determined at three different substrate concentrations, and is measured as the change in optical absorbance at 405 nm.
Test compound solutions (100 L; normally in buffer or saline containing BSA 10 g/L) are mixed with 200 L of human -thrombin (Sigma Chemical Co) in assay buffer (0.05 mol/L Tris-HCl pH 7.4, ionic strength 0.15 adjusted with NaCl) containing BSA (10 g/L), and analysed as samples in the Cobas Bio. A 60 L sample, together with 20 L of water, is added to 320 L of the substrate S-2238 (Chromogenix AB, Mxc3x6lndal, Sweden) in assay buffer, and the absorbance change (A/min) is monitored. The final concentrations of S-2238 are 16, 24 and 50 xcexcmol/L and of thrombin 0.125 NIH U/mL.
The steady state reaction rate is used to construct Dixon plots, i.e. diagrams of inhibitor concentration vs. 1/(A/min). For reversible, competitive inhibitors, the data points for the different substrate concentrations typically form straight lines which intercept at x=-Ki.
Test D
Determination of Activated Partial Thromboplastin Time (APTT)
APTT is determined in pooled normal human citrated plasma with the reagent PTT Automated 5 manufactured by Stago. The inhibitors are added to the plasma (10 L inhibitor solution to 90 L plasma) and incubated with the APTT reagent for 3 minutes followed by the addition of 100 L of calcium chloride solution (0.025 M) and APTT is determined by use of the coagulation analyser KC10 (Amelung) according to the instructions of the reagent producer.
The clotting time is expressed as absolute values (seconds) as well as the ratio of APTT without inhibitor (APTT0) to APTT with inhibitor (APTT1). The latter ratios (range 1-0) are plotted against the concentration of inhibitor (log transformed) and fitted to sigmoidal dose-response curves according to the equation
y=a/[1+(x/IC50)s]
where: a=maximum range, i.e. 1; s=slope of the dose-response curve; and IC50=the concentration of inhibitor that doubles the clotting time. The calculations are processed on a PC using the software program GraFit Version 3, setting equation equal to: Start at 0, define end=1 (Erithacus Software, Robin Leatherbarrow, Imperial College of Science, London, UK). IC50APTT is defined as the concentration of inhibitor in human plasma that doubled the Activated Partial Thromboplastin Time.
Test E
Determination of Thrombin Time ex vivo
The inhibition of thrombin after oral or parenteral administration of the compounds of formula I, dissolved in ethanol:Solutol3:water (5:5:90), is examined in conscious rats which, one or two days prior to the experiment, are equipped with a catheter for blood sampling from the carotid artery. On the experimental day blood samples are withdrawn at fixed times after the administration of the compound into plastic tubes containing 1 part sodium citrate solution (0.13 mol per L) and 9 parts of blood. The tubes are centrifuged to obtain platelet poor plasma. The plasma is used for determination of thrombin time or ecarin clotting time (ECT) as described below.
The citrated rat plasma, 100 L, is diluted with a saline solution, 0.9%, 100 L, and plasma coagulation is started by the addition of human thrombin (T 6769, Sigma Chem Co, USA or Hematologic Technologies) in a buffer solution, pH 7.4, 100 L, or ecarin (Pentapharm). The clotting time is measured in an automatic device (KC 10, Amelung, Germany).
Where a xe2x80x9cprodrugxe2x80x9d compound of formula I (e.g. of formula IA) is administered, concentrations of the appropriate active thrombin inhibitor of formula I in the rat plasma are estimated by the use of standard curves relating the thrombin time or ecarin clotting time in the pooled citrated rat plasma to known concentrations of the corresponding xe2x80x9cactivexe2x80x9d thrombin inhibitor dissolved in saline.
Based on the estimated plasma concentrations of the active thrombin inhibitor (which assumes that thrombin time or ECT prolongation is caused by the aforementioned compound) in the rat, the area under the curve after oral and/or parenteral administration of the corresponding prodrug compound of formula I is calculated (AUCpd) using the trapezoidal rule and extrapolation of data to infinity.
The bioavailability of the active thrombin inhibitor after oral or parenteral administration of the prodrug is calculated as below:
[(AUCpd/dose)/(AUCactive,parenteral/dose]xc3x97100
where AUCactive,parenteral represents the AUC obtained after parenteral administration of the corresponding active thrombin inhibitor to conscious rats as described above.
Test F
Determination of Thrombin Time in Urine ex vivo
The amount of the xe2x80x9cactivexe2x80x9d thrombin inhibitor that is excreted in urine after oral or parenteral administration of xe2x80x9cprodrugxe2x80x9d compounds of the invention, dissolved in ethanol:Solutol3:water (5:5:90), is estimated by determination of the thrombin time in urine ex vivo (assuming that thrombin time prolongation is caused by the aforementioned compound). Conscious rats are placed in metabolism cages, allowing separate collection of urine and faeces, for 24 hours following oral administration of compounds of the invention. The thrombin time is determined on the collected urine as described below.
Pooled normal citrated human plasma (100 L) is incubated with the concentrated rat urine, or saline dilutions thereof, for one minute. Plasma coagulation is then initiated by the administration of human thrombin (T 6769, Sigma Chem Company) in buffer solution (pH 7.4; 100 L). The clotting time is measured in an automatic device (KC 10; Amelung).
The concentrations of the active thrombin inhibitor in the rat urine are estimated by the use of standard curves relating the thrombin time in the pooled normal citrated human plasma to known concentrations of the aforementioned active thrombin inhibitor dissolved in concentrated rat urine (or saline dilutions thereof). By multiplying the total rat urine production over the 24 hour period with the estimated mean concentration of the aforementioned active inhibitor in the urine, the amount of the active inhibitor excreted in the urine (AMOUNTpd) can be calculated.
The bioavailability of the active thrombin inhibitor after oral or parenteral administration of the prodrug is calculated as below:
[(AMOUNTpd/dose)/(AMOUNTactive,parenteral/dose]xc3x97100
where AMOUNTactive,parenteral represents the amount excreted in the urine after parenteral administration of the corresponding active thrombin inhibitor to conscious rats as described above.
Test G
Metabolic Activation of Prodrug Compounds in vitro
Prodrug compounds of formula IA are incubated at 37xc2x0 C. with liver microsomes or 10 000 g (referring to the centrifuge speed) supernatant fractions (i.e. s9 fraction) prepared from human or rat liver homogenate. The total protein concentration in the incubations are 1 or 3 mg/mL dissolved in 0.05 mol/L TRIS buffer (pH 7.4), and with the cofactors NADH (2.5 mmol/L) and NADPH (0. 8 mmol/L) present. The total volume of the incubate is 1.2 mL. The initial prodrug concentrations are 5 or 10 mol/L. Samples are collected from the incubate at regular is intervals more than 60 minutes after the start of the incubations. Samples (25 L) from the incubate are mixed with an equal volume of human or rat plasma and an appropriate amount of thrombin, and the clotting time (i.e. thrombin time) is measured on a coagulometer (KC 10; Amelung). The amount of xe2x80x9cactivexe2x80x9d thrombin inhibitor formed is estimated by the use of standard curves relating the thrombin time in pooled citrated human or rat plasma to known concentrations of the corresponding xe2x80x9cactive thrombin inhibitorxe2x80x9d.
The amount of xe2x80x9cactivexe2x80x9d thrombin inhibitor is alternatively, or in addition to the above-mentioned method, estimated by the use of LC-MS.
Test H
Determination of Plasma Clearance in Rat
Plasma clearance was estimated in male Sprague Dawley rats. The compound was dissolved in water and administered as a subcutaneous bolus injection at a dose of 4 xcexcmol/kg. Blood samples were collected at frequent intervals up to 5 hours after drug administration. Blood samples were centrifuged and plasma was separated from the blood cells and transferred to vials containing citrate (10% final concentration). The ecarin clotting time (ECT) was then determined in each plasma sample by the use of a coagulometer (KC10; Amelung). The plasma concentration in each sample was determined by the use of a standard curve relating the ECT in pooled citrated plasma samples to known concentrations of the compound. The area under the plasma concentration-time profile was estimated using the log/linear trapezoidal rule and extrapolated to infinite time. Plasma clearance (CL) of the compound was then determined as
CL=Dose/AUC
The values are reported in mL/min/kg.
Test I
Determination of in vitro Stability of Active Thrombin Inhibitors
Liver microsomes were prepared from Sprague-Dawley rats and human liver samples according to internal SOPs. The compounds were incubated at 37xc2x0 C. at a total microsome protein concentration of 3 mg/mL in a 0.05 mol/L TRIS buffer at pH 7.4, in the presence of the cofactors NADH (2.5 mmol/L) and NADPH (0.8 mmol/L). The initial concentration of compound was 5 or 10 xcexcmol/L. Samples were taken for analysis up to 60 minutes after the start of the incubation. The enzymatic activity in the collected sample was immediately stopped by adding 20% myristic acid at a volume corresponding to 3.3% of the total sample volume. The concentration of compound remaining (FINAL CONC) in the 60 min. sample was determined by means of LCMS using a sample collected at zero time as reference (START CONC). The % of degraded thrombin inhibitor was calculated as:   100  ⁢  %  xc3x97                    [START  CONC]            -              [FINAL  CONC]                    [START  CONC]      
Test J
Arterial Thrombosis Model
Vessel damage was induced by applying ferric chloride (FeCl3) topically to the carotid artery. Rats are anaesthetised with an intraperitoneal injection of sodium pentobarbital (80 mg/kg; Apoteksbolaget; Umea, Sweden), followed by continuous infusion (12 mg/kg/h) throughout the experiment. Rat body temperature was maintained at 38xc2x0 C. throughout the experiment by external heating. The experiment started with a 5 minutes control period. Five minutes later, human 125I-fibrinogen (80 kBq; IM53; Amersham International, Buckinghamshire, UK) was given intravenously and was used as a marker for the subsequent incorporation of fibrin(ogen) into the thrombus. The proximal end of the carotid artery segment was placed in a plastic tube (6 mm; Silastic(copyright); Dow Corning, Mich., USA) opened lengthways, containing FeCl3-soaked (2 xcexcl; 55% w/w; Merck, Darmstadt, Germany) filter paper (diameter 3 mm; IF; Munktell, Grycksbo, Sweden). The left carotid artery was exposed to FeCl3 for 10 minutes and was then removed from the plastic tube and soaked in saline. Fifty minutes later, the carotid artery was removed and rinsed in saline. Reference blood samples were also taken for determination of blood 125I-activity, 10 minutes after the injection of 125I -fibrinogen, and at the end of the experiment. The 125I-activity in the reference blood samples and the vessel segment were measured in a gamma counter (1282 Compugamma; LKB Wallac Oy, Turku, Finland) on the same day as the experiment was performed. The thrombus size was determined as the amount of 125I-activity incorporated in the vessel segment in relation to the 125I-activity in the blood (cpm/mg).